Novel Translational Regulation of the Proapoptotic bcl2 Member Puma and Its Role During Skeletal Myoblast Apoptosis

Differentiation and apoptosis are coordinately regulated in skeletal myoblasts. During the process of skeletal myoblast differentiation, our lab has determined that roughly 30 of myoblasts undergo apoptosis rather than differentiation. Further, we have reported that cytochrome C is released, and that the expression level of the pro-apoptotic Bcl2 family member PUMA is elevated, when 23A2 myoblasts are cultured in DM. Our long-term goal is to identify targets for manipulation that would abrogate this apoptosis without affecting differentiation. These findings could be significant to the study of myoblast transfer as a therapeutic approach. The goals of this dissertation are to: 1- determine the significance of PUMA in apoptosis associated with skeletal myoblast differentaion 2- determine the mechanism responsible for the increased expression of PUM

Novel Translational Regulation of the Proapoptotic bcl2 Member Puma and Its Role During Skeletal Myoblast Apoptosis

Differentiation and apoptosis are coordinately regulated in skeletal myoblasts. During the process of skeletal myoblast differentiation, our lab has determined that roughly 30 of myoblasts undergo apoptosis rather than differentiation. Further, we have reported that cytochrome C is released, and that the expression level of the pro-apoptotic Bcl2 family member PUMA is elevated, when 23A2 myoblasts are cultured in DM. Our long-term goal is to identify targets for manipulation that would abrogate this apoptosis without affecting differentiation. These findings could be significant to the study of myoblast transfer as a therapeutic approach. The goals of this dissertation are to: 1- determine the significance of PUMA in apoptosis associated with skeletal myoblast differentaion 2- determine the mechanism responsible for the increased expression of PUM

Novel Translational Regulation of the Proapoptotic bcl2 Member Puma and Its Role During Skeletal Myoblast Apoptosis

Differentiation and apoptosis are coordinately regulated in skeletal myoblasts. During the process of skeletal myoblast differentiation, our lab has determined that roughly 30 of myoblasts undergo apoptosis rather than differentiation. Further, we have reported that cytochrome C is released, and that the expression level of the pro-apoptotic Bcl2 family member PUMA is elevated, when 23A2 myoblasts are cultured in DM. Our long-term goal is to identify targets for manipulation that would abrogate this apoptosis without affecting differentiation. These findings could be significant to the study of myoblast transfer as a therapeutic approach. The goals of this dissertation are to: 1- determine the significance of PUMA in apoptosis associated with skeletal myoblast differentaion 2- determine the mechanism responsible for the increased expression of PUM

Aberrant iPSC-derived human astrocytes in Alzheimer's disease

The pathological potential of human astroglia in Alzheimer's disease (AD) was analysed in vitro using induced pluripotent stem cell (iPSC) technology. Here, we report development of a human iPSC-derived astrocyte model created from healthy individuals and patients with either early-onset familial AD (FAD) or the late-onset sporadic form of AD (SAD). Our chemically-defined and highly efficient model provides >95% homogeneous populations of human astrocytes within 30 days of differentiation from cortical neural progenitor cells (NPCs). All astrocytes expressed functional markers including; glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 1 (EAAT1), S100B and glutamine synthetase (GS) comparable to that of adult astrocytes in vivo. However, induced astrocytes derived from both SAD and FAD patients exhibit a pronounced pathological phenotype, with a significantly less complex morphological appearance, overall atrophic profiles, and abnormal localisation of key functional astroglial markers. Furthermore, NPCs derived from identical patients did not show any differences, therefore, validating that remodelled astroglia are not as a result of defective neuronal intermediates. This work not only presents a novel model to study the mechanisms of human astrocytes in vitro, but also provides an ideal platform for further interrogation of early astroglial cell-autonomous events in AD and the possibility of identification of novel therapeutic targets for the treatment of AD

Stem cell-derived astrocytes:are they physiologically credible?

Astrocytes are now increasingly acknowledged as having fundamental and sophisticated roles in brain function and dysfunction. Unravelling the complex mechanisms that underlie human brain astrocyte-neuron interactions is therefore an essential step on the way to understanding how the brain operates. Insights into astrocyte function to date, have almost exclusively been derived from studies conducted using murine or rodent models. Whilst these have led to significant discoveries, preliminary work with human astrocytes has revealed a hitherto unknown range of astrocyte types with potentially greater functional complexity and increased neuronal interaction with respect to animal astrocytes. It is becoming apparent, therefore, that many important functions of astrocytes will only be discovered by direct physiological interrogation of human astrocytes. Recent advancements in the field of stem cell biology have provided a source of human based models. These will provide a platform to facilitate our understanding of normal astrocyte functions as well as their role in CNS pathology. A number of recent studies have demonstrated that stem cell derived astrocytes exhibit a range of properties, suggesting that they may be functionally equivalent to their in vivo counterparts. Further validation against in vivo models will ultimately confirm the future utility of these stem-cell based approaches in fulfilling the need for human- based cellular models for basic and clinical research. In this review we discuss the roles of astrocytes in the brain and highlight the extent to which human stem cell derived astrocytes have demonstrated functional activities that are equivalent to that observed in vivo

Increased Expression of the Pro-Apoptotic Bcl2 Family Member PUMA and Apoptosis by the Muscle Regulatory Transcription Factor MyoD in Response to a Variety of Stimuli

We have previously reported that the level of MyoD expression correlates with the level of apoptosis that occurs in a subpopulation of skeletal myoblasts induced to differentiate by serum withdrawal. Herein we document that MyoD expression contributes to the level of apoptosis in myoblasts and fibroblasts in response to a variety of apoptotic stimuli. Specifically, re-expression of MyoD in skeletal myoblasts rendered defective for both differentiation and apoptosis by the expression of oncogenic Ras restores their ability to undergo both differentiation and apoptosis in response to serum withdrawal. Further, using a fibroblast cell line expressing an estrogen receptor:MyoD fusion protein, we have determined that addition of estrogen sensitizes these fibroblasts to apoptosis induced by serum withdrawal, or by treatment with etoposide or thapsigargin. RNAi mediated silencing of MyoD in either 23A2 or C2C12 myoblasts renders these cells resistant to apoptosis induced by serum withdrawal, or by treatment with etoposide or thapsigargin. Finally, MyoD mediated regulation of the apoptotic response to these various stimuli, in both myoblasts and fibroblasts, correlates with the level of induction of the pro-apoptotic Bcl2 family member PUMA. © 2009 Springer Science+Business Media, LLC

Increased Expression of the Pro-Apoptotic Bcl2 Family Member PUMA Is Required for Mitochondrial Release of Cytochrome C and the Apoptosis Associated With Skeletal Myoblast Differentiation

We have previously shown that when skeletal myoblasts are cultured in differentiation medium (DM), roughly 30% undergo caspase 3-dependent apoptosis rather than differentiation. Herein, we investigate the molecular mechanism responsible for the activation of caspase 3 and the ensuing apoptosis. When 23A2 myoblasts are cultured in DM, caspase 9 activity is increased and pharmacological abrogation of caspase 9 activation impairs caspase 3 activation and apoptosis. Further, we detect a time dependent release of mitochondrial cytochrome C into the cytosol in roughly 30% of myoblasts. Inclusion of cycloheximide inhibits the release of cytochrome C, the activation of caspase 9 and apoptosis. These data indicate that the mitochondrial pathway plays a role in this apoptotic process and that engagement of this pathway relies on de novo protein synthesis. Through RT-PCR and immunoblot analysis, we have determined that the expression level of the pro-apoptotic Bcl2 family member PUMA is elevated when 23A2 myoblasts are cultured in DM. Further, silencing of PUMA inhibits the release of cytochrome C and apoptosis. Signaling by the transcription factor p53 is not responsible for the increased level of PUMA. Finally, myoblasts rescued from apoptosis by either inhibition of elevated caspase 9 activity or silencing of PUMA are competent for differentiation. These results indicate a critical role for PUMA in the apoptosis associated with skeletal myoblast differentiation and that a p53-independent mechanism is responsible for the increased expression of PUMA in these cells. © 2007 Springer Science+Business Media, LLC

Mitochondrial Alterations by PARKIN in Dopaminergic Neurons Using PARK2 Patient-Specific and PARK2 Knockout Isogenic iPSC Lines

In this study, we used patient-specific and isogenic PARK2-induced pluripotent stem cells (iPSCs) to show that mutations in PARK2 alter neuronal proliferation. The percentage of TH+ neurons was decreased in Parkinson’s disease (PD) patient-derived neurons carrying various mutations in PARK2 compared with an age-matched control subject. This reduction was accompanied by alterations in mitochondrial:cell volume fraction (mitochondrial volume fraction). The same phenotype was confirmed in isogenic PARK2 null lines. The mitochondrial phenotype was also seen in non-midbrain neurons differentiated from the PARK2 null line, as was the functional phenotype of reduced proliferation in culture. Whole genome expression profiling at various stages of differentiation confirmed the mitochondrial phenotype and identified pathways altered by PARK2 dysfunction that include PD-related genes. Our results are consistent with current model of PARK2 function where damaged mitochondria are targeted for degradation via a PARK2/PINK1-mediated mechanism